METHODS: Mixtures of
plasmids and serum samples from 52 chronic hepatitis B patients with
low abundant lamivudine-resistant mutations were tested with LDR and
real-time PCR. Time required and reagent cost for both assays were
evaluated.

RESULTS: Real-time PCR
detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR
detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures
with YMDD plasmid of 106 copies/mL. Among the 52 clinical
serum samples, completely concordant results were obtained for all
samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were
detected. Cost and time required for LDR and real-time PCR are 60/80
CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.

CONCLUSION: LDR and
real-time PCR are both sensitive and inexpensive methods for
monitoring low abundant YMDD mutants during lamivudine therapy in
patients with chronic hepatitis B. LDR is more sensitive and less
expensive, while real-time PCR is more rapid.

Lamivudine is an effective antiviral agent for
treatment of patients with chronic hepatitis B and advanced liver
diseases[1]. However, long-term lamivudine monotherapy
leads to emergence of lamivudine-resistant hepatitis B virus (HBV)
mutants in some patients chronically infected with HBV[1,2].
The incidence is 16%-32% in the first year and increases to 38%,
57%, and 67% after 2, 3, and 4 years, respectively[3-6].
Resistance is associated with mutations in the highly conserved
tyrosine-methionine-aspartate-aspartate (YMDD) motif of the reverse
transcriptase, which is part of the catalytic site of the HBV
polymerase[7]. Viological breakthrough and alanine
transaminase (ALT) elevation have been shown to occur 2-28 wk and
12-31 wk after the emergence of YMDD mutants, respectively[8-10].
Initially, YMDD mutants consist of minor populations. They gradually
replace the wild-type virus, reaching a 100% lamivudine-resistant
variant population, and this replacement occurs in parallel with the
increase in HBV DNA load[10]. Sensitive methods for early
detection of lamivudine-resistant mutants will be helpful for
physicians to make clinical decisions in treatment of patients with
HBV infection.

Several technologies have been developed for the
detection of lamivudine-resistant mutants[11]. Although
nucleotide sequencing of PCR products is widely used to detect
lamivudine resistance, it is expensive and laborious, and can detect
only mutant viruses representing at least 50% of the total virus
population[8]. Inno-LiPA, pyrosequencing, real-time PCR,
and ligase detection reaction (LDR) are able to detect low abundant
YMDD mutants in the wild-type HBV[8,12-15]. However, only
few studies have compared these methods.

We have previously compared real-time PCR and
pyrosequencing for detection of YMDD mutants in patients with
chronic hepatitis B[16]. In the present study, we
compared LDR and real-time PCR for detection of low abundant YMDD
mutants in mixed plasmids and clinical samples from lamivudine
treated patients with chronic hepatitis B.

MATERIALS AND METHODS

Plasmids and controls

Plasmids and controls were prepared as previously
described[14,16]. In brief, three previously identified
serum samples containing HBV with YMDD, YVDD and YIDD sequences were
used as template and amplified by PCR. PCR products were cloned
using pGEM-T systems (Promega, Madison, Wisconsin, USA), and clones
were sequenced using ABI 3100 sequencer (Applied Biosystems, Foster,
California, USA).

Patients and samples

Serum samples were collected from 196 patients with
chronic HBV infection. All patients were treated with lamivudine for
three months to three years and serum HBV-DNA levels were above 1.0
× 104 copies/mL by real-time PCR. Among these samples, 52
samples with YMDD mutants below 50% of total HBV population
(determined by real-time PCR[14]) were selected for
comparison of LDR and real-time PCR. All these 52 samples were found
to contain only the YMDD variant by sequencing of PCR products, but
found to contain YVDD or YIDD variants with real-time PCR or LDR.

Extraction and quantitation of HBV DNA

HBV DNA was extracted from serum samples using the
HBV DNA extraction reagents (Fosun Diagnostics, Shanghai, China)
according to the manufacturer’s instructions. Serum HBV DNA levels
were measured on ABI 7300 real-time PCR system (Applied Biosystems,
Foster, California, USA) with quantitative real-time PCR reagents (Fosun
Diagnostics, Shanghai, China), which was approved by the State Food
and Drug Administration of China for in vitro diagnostic use.

Sequencing of PCR products

HBV DNA samples were prepared for sequencing by
amplification with PCR as described by Allen et al[7].
HBV DNA extracted from serum samples was amplified by PCR. PCR
products were purified with QIAquick PCR purification kits (Qiagen,
Chatsworth, California, USA) and were eluted from the column with 80
mL of distilled deionized water. The DNA quality and concentration
were determined by absorbance measurements at 260 and 280 nm and by gel
electrophoresis on a 2.5% agarose gel. All sequencing reactions were
performed on ABI 3130 DNA sequencer (Applied Biosystems, Foster,
California, USA).

Ligase detection reaction

LDR was carried out as described by Xiao et al[15].
In brief, for one type of mutant (YIDD or YVDD), one common probe
and two discriminating probes for mutant and wild-type YMDD were
used in LDR, which was carried out in 20 mL of buffer, 1 pmol of
each probe, and 5 mL of sample DNA. The reaction mixture was
incubated at 94℃ for 2 min, before adding 15 U of thermostable Taq
DNA ligase (New England Biolabs, USA), followed by 20 cycles of 30 s
at 94℃ and 4 min at 65℃. Two PCR reactions were performed with the
product of the LDR as template for 30 cycles at 94℃ for 30 s, 60℃
for 30 s, and 72℃ for 45 s. The PCR products were separated by
agarose gel electrophoresis and visualized with ethidium bromide
staining.

Real-time PCR

Real-time PCR for detection of YMDD mutants was
performed as previously described[14,16]. In brief,
parallel reaction C, V and I were used to detect total HBV, YVDD and
YIDD variants, respectively. The amplification was performed on ABI
7300 PCR system (Applied Biosystems,
Foster, CA, USA) by incubating the
reaction mixture (50 mL) at 50 degree for two minutes,
followed by 5 min at 95 degree, 40 cycles of PCR amplification (94
degree for 20 s and 53 degree for 30 s). The reaction system was
provided and optimized by Fosun Diagnostics (Fosun Diagnostics,
Shanghai, China). The percentage of mutants in total virus was
calculated by the following equations[14]:

(1) DCt = Ct of control - Ct of mutants

(2) Ratio of mutants to total virus = 2ÄΧt

Mixing experiments

Mixing experiments were carried out as previously
described[16]. In brief, mutant plasmid containing YIDD
sequence and wild-type plasmid were mixed at a final concentration
of 106 copies/mL, and the percentage of the YIDD plasmid
in the mixture was 100%, 50%, 10%, 1%, 0.1%, and 0.01%,
respectively. The mixtures were analyzed by LDR and real-time PCR
respectively. For real-time PCR, each mixture was analyzed five
times, and the mean Ct value of the five runs was used to determine
the ratio of mutant to total viruses. For LDR assay, each mixture
was analyzed only once.

Time study

Two skilled technicians were selected to perform the
assays. Time required for each assay was measured by direct
observation during the procedures performed by the technicians,
including the process of DNA extraction, amplification, detection,
and analysis.

Cost analysis

Cost for each assay was estimated based on the prices
of reagents in China. The cost of instruments and labors was not
included.

RESULTS

Detection of mixed plasmids

Mixtures of plasmids contained YIDD and YMDD at
different ratios were detected by LDR and real-time PCR,
respectively. LDR detected YIDD, YIDD/YMDD in the mixtures
containing 100%, 50%, 10%, 1%, 0.1% and 0.01% YIDD plasmid.
Real-time PCR detected YIDD in the mixture containing 100% YIDD
plasmid and YIDD/YMDD in the mixtures containing 50%, 10%, 1% and
0.1% YIDD plasmids, but detected only YMDD in the mixture containing
0.01% YIDD plasmid (Table 1). It means that real-time PCR and
LDR are able to detect 1000 and 100 copies/mL of mutant virus in the
background of wild type viruses, respectively. The results of
real-time PCR were consistent with our previous study with mixtures
of YVDD and YMDD plasmids[16].

Comparison of LDR and real-time PCR for
detection of clinical samples with low abundant YMDD mutants

We tested clinical serum samples from 52 lamivudine
treated patients with chronic hepatitis B who had low abundant YMDD
mutants. All the samples were detected as YMDD virus by sequencing
the PCR products. The results obtained by LDR and real-time PCR were
consistent (Table 2). Both methods detected 39 YIDD, 9 YVDD,
and 4 YIDD/YVDD. The percentages of mutants in the virus population
obtained by real-time PCR ranged from 4% to 40%. The percentage of
the four YIDD/YVDD mixed mutants was 10%/20%, 30%/20%, 40%/10%, and
20%/30%, respectively.

Time required

In this study, we used 96-well PCR equipment and all
the 52 samples were dealt with in a run. The total assay time for
LDR and real-time PCR was 4.5 and 2.5 h, respectively.

Cost

The cost per test for each assay was calculated based
on the prices of the reagents in China. Primers and probe were
synthesized in TaKaRa Biotech (TaKaRa, Dalian, China). Real-time PCR
mixtures were from Fosun Diagnostics (Fosun Diagnostics, Shanghai,
China). The total reagent cost per test for LDR and real-time PCR
was 60 and 80 CNY (8 and 10.7 US dollars), respectively. Although
the cost of labors is similar in the same region, the cost of
equipment used for LDR assay is much lower than that for PCR assay
(5000 US dollars vs 60 000 US dollars).

DISCUSSION

Lamivudine has revolutionized the treatment of
chronic hepatitis B. Lamivudine-resistant mutations in the YMDD
motif of polymerase gene were detected in lamivudine treated and
untreated patients with chronic hepatitis B[14,17-19].
Clinical breakthrough was observed 2 wk-7 mo after the emergence of
YMDD mutations[8-10], causing considerable morbidity and
mortality in those patients[20-24]. Lamivudine-resistant
mutants are frequently preexisting variants in HBV-infected patients
and are selected during lamivudine therapy. These resistant variants
initially represent a minority of the quasispecies and gradually
replace the wild-type YMDD variants[10]. Detection of low
abundant lamivudine-resistant mutants in the background of wild-type
HBV as early as possible is helpful for virological follow-up and
diagnosis of resistance in the clinical setting.

To date, many assays have been used for detection of
lamivudine-resistant mutants in patients with hepatitis B[11].
The differences in sensitivity, specificity, cost, and time required
do exist in these methods. Real-time PCR is able to quantitatively
detect a small portion of resistant mutants in HBV populations and
LDR is a newly developed method for detection of low abundant
mutants in the background of wild-type HBV. In the present study, we
compared LDR and real-time PCR for detection of low abundant YMDD
mutations in lamivudine treated patients. The results obtained by
the two methods were completely concordant in all samples, and 39
YIDD, 9 YVDD, and 4 YIDD/YVDD variants were detected. The
percentages of mutants in the virus population obtained by real-time
PCR ranged from 4% to 40%. In the mixing experiment, LDR was able to
detect as low as 0.01% (100 copies/mL) of YIDD plasmid, while
real-time PCR only detected 0.1% (1000 copies/mL) of YIDD plasmid in
the background of YMDD plasmid. This may be due to LDR employing two
kinds of amplification cycles, 20 cycles of LDR and 30 cycles of PCR,
in the testing process. These results suggest that LDR is more
sensitive than real-time PCR. In addition, the cost of LDR is
slightly lower than that of real-time PCR. However, real-time PCR is
much more rapid and requires less manual work than LDR. Both methods
are sensitive and inexpensive compared to other methods for
detection of YMDD mutation[16]. Another advantage of the
real-time PCR method is that it is able to calculate the ratio of
mutants to total virus in samples[14]. This will be
useful in the clinical studies on the dynamics of resistant mutants
during lamivudine therapy.

Several antiviral agents, such as adefovir and
entecavir, can provide effective therapies in patients with
lamivudine-resistant HBV[25-27]. Pegylated interferon
also induces sustained responses in a portion of lamivudine-resistant
patients[28-30]. Monitoring low abundant YMDD mutation
during lamivudine therapy by sensitive and inexpensive methods will
be helpful for physicians to make better clinical decisions as early
as possible in management of chronic hepatitis B.

In conclusion, both LDR and real-time PCR are
sensitive and inexpensive methods for monitoring low abundant YMDD
mutations during lamivudine therapy in patients with chronic
hepatitis B. LDR is more sensitive and less expensive, while
real-time PCR is more rapid.

COMMENTS

Background

Many assays have been used for detection of
lamivudine-resistant mutants in patients with hepatitis B. The
differences in sensitivity, specificity, and cost do exist in these
methods. However, only a few studies have compared these methods.

Research frontier

Lamivudine-resistant variants initially represent a
minority of the viruses and gradually replace the wild-type YMDD
variants. Methods for detection of low abundant lamivudine-resistant
mutants in the background of wild-type hepatitis B virus (HBV) as
early as possible are helpful for diagnosis of resistance in the
clinical setting.

Related publications

Shi et al
and Xiao et al
developed real-time PCR and LDR assays for detection of minority
lamivudine-resistant mutants in patients with hepatitis B. However,
they did not compare the clinical performance between the two
methods.

Innovations and breakthroughs

This article compared LDR and real-time PCR for
detection of low abundant YMDD mutations in lamivudine treated
patients. Both assays are sensitive and inexpensive for monitoring
low abundant YMDD mutations during lamivudine therapy in patients
with chronic hepatitis B. LDR is more sensitive and less expensive,
while real-time PCR is more rapid.

Applications

Both LDR and real-time PCR are suitable for early
detection of lamivudine-resistant mutations in patients treated with
lamivudine.

Terminology

Ligase detection reaction (LDR) detects nucleotide
sequence by annealing and subsequent ligation of two
oligonucleotides (probe and detector). Ligation of the probe and
detector occurs only when the two bases on either side of the
ligation site are complementary to the template. LDR is usually
coupled with PCR for detection of low abundant point mutations.

Peer review

This study is of importance for the early detection
of lamivudine-resistant HBV mutants in patients with chronic HBV
infection. The experiments appear to be conducted very carefully and
by an experienced team of investigators.